Fulvic acid and its use in the treatment of various conditions

ABSTRACT

Fulvic acid salts, esters or derivatives thereof in pharmaceutical preparations are effective for treating inflammation, acne, exzema or bacterial or fungal or viral infections. These pharmaceutical preparations may be given either orally or topically in the form of a solution, paste, ointment, powder to humans or animals.

This application is the national phase under 35 U.S.C. §371 of PCTInternational Application No. PCT/IB99/01649 which has an Internationalfiling date of Oct. 8, 1999, which designated the United States ofAmerica.

FIELD OF THE INVENTION

This invention relates to fulvic acid and its use in the treatment ofvarious conditions.

Humic substances are ubiquitous in nature and arise from the decay ofplant and animal residues in the environment (MacCarthe et al. 1990).These substances can be divided into humic acid, fulvic acid and humanon the basis of the solubility in water as a function of pH. Fulvic acidis the fraction that is soluble in water under all pH conditions and isin general lower in molecular size and weight and lower in colourintensity than humic acids.

Humic substances commonly account for 50% of the dissolved organiccarbon concentrations in stream water, of which 90 to 95% are fulvicacids. Humic acids are 3 to 5 times more abundant in soils than fulvicacids (Stevenson, 1982), whereas fulvic acids are 9 to 10 times moreabundant in water than humic acids (Malcolm, 1985).

Humic acids have been successfully used in the treatment of:

(i) hyperacidity and other gastric disturbances in humans (Reichert,1966, Gramsch, 1961)

(ii) inflammation (Salz 1974, Motohisa et al., 1974)

(iii) anemia and hypercholesterolemia (Soloveyva and Lotosh 1984)

(iv) Von Willebrand disease (Lopez-Fernandez et al., 1992)

The possible application of fulvic acid in the treatment of human andanimal diseases has, up to now, not been investigated.

Wang et al (1996) studied the interaction between fulvic acids andactive oxygen free radical and found fulvic acids from peat were able toscavenge both superoxide and hydroxyl radical. It has also been shownthat fulvic acids prevent the absorption of mutagens through the ratsmall intestine using a highly mutagenic furanone found in chlorinatedwater and an in vitro everted rat gut sac system (Clark and Chipman,1995).

Although the presence of fulvic acids in the drinking water of certainparts of China has been coupled to the incidence of Kashin-Beck disease,this only occurred in conjunction with a selenium deficient diet (Pengand Xu, 1987).

U.S. Pat. Nos. 4,999,202 and 5,204,368 disclose compositions havingbacterial and bacteriostatic properties containing a fulvic acid, saltor derivative thereof as the active ingredient. These compositions aredescribed as being useful as disinfectants.

SUMMARY OF THE INVENTION

According to one aspect of the invention, there is provided apharmaceutical composition comprising a fulvic acid, salt, ester orderivative thereof as the active ingredient.

The pharmaceutical composition may be provided for oral or topicaladministration to a subject.

In the case of topical administration, the composition may be providedin the form of a solution, paste, ointment, powder or any other formsuitable for topical administration.

The subject may be a human or an animal.

Further according to the invention, there is provided the use of afulvic acid, salt, ester or derivative thereof, in the treatment of acondition of a subject. The condition may, for example, be inflammation,acne, eczema or bacterial or fungal or viral infections.

The treatment of the condition may be by oral or topical administration.

The subject is typically a human or an animal.

The fulvic acid is preferably a fulvic acid derived from a wet coaloxidation process of the type described in U.S. Pat. No. 4,912,256. Sucha fulvic acid is hereinafter referred to as “oxifulvic acid or OFA”.

Bergh et al. (1997) identified almost 50 different compounds, most ofwhich were carboxylic acids, in oxifulvic acid. The compounds weremostly ordinary physiological metabolites with no evidence of any toxiccompound in the product mixture.

A typical functional group analysis of oxifulvic acid is given below:

Total acid groups: 11.5-15.5 meq/g

Carboxylic groups: 8.5-12.5 meq/g

Phenolic groups: 2.3-3.7 meq/g

In an example of the invention, a composition for topical application toa human or animal Comprises 4.5 percent, or 9.0 percent, by mass ofoxifulvic acid in an aqueous cream.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1 to 4 illustrate graphically the results of certain tests carriedout on oxifulvic acid, and

FIGS. 5A and 5B illustrate photographically the results of treatingpyotraumatic dermatitis using an oxifulvic acid cream of the invention.

DESCRIPTION OF EMBODIMENTS

The active ingredient in the practice of the invention is a fulvic acid,salt, ester or derivative thereof. Oxifulvic acid, having the preferredfunctional group analysis mentioned above, has been subjected to anumber of in vitro and in vivo studies and these are describedhereinafter.

The Effects of Oxifulvic Acid on Immune Functions In Vitro

The Effects of Oxifulvic Acid on the Production of Oxidants by HumanNeutrophils.

Human neutrophils (separated on Ficoll) were treated with oxifulvic acid(at 12.5 and 25 ug/ml) for 15 min at 37° C. The cells were stimulatedwith PMA (phorbol myristate acetate) (20 ng/ml) and superoxideproduction determined by measuring the superoxide-inhibitable reductionof ferricytochrome C (1 mM). A significant inhibition of superoxideproduction was observed at both concentrations tested (Table 1).

TABLE 1 The effects of oxifulvic acid on PMA-activated neutrophilsuperoxide production nmoles superoxide/ Treatment 10⁶ neutrophils/10min* Resting Control 0.072 Stimulated (+PMA) Control 67.2 + oxifulvicacid (12.5 ug/ml) 59 (25 ug/ml) 43 *average data of two experiments

The superoxide scavenging activities of oxifulvic acid (at 25 ug/ml)were determined using the hypoxanthine (1 mM)/xanthine-oxidase (100mU/ml) enzymatic system to generate superoxide. Oxifulvic acid exhibitedsuperoxide-scavenging activity (Table 2).

TABLE 2 Superoxide scavenging activities of oxifulvic acid at 25 ug/ml.Treatment nmoles superoxide* Control 55.95 Oxifulvic acid 41.25 *averagedata of two experiments

The Effects of Oxifulvic Acid on the Proliferative Response of a MixedLymphocyte Culture

These experiments were performed by co-culturing the lymphocytes of twodifferent donors in both the presence and absence of serial dilutions ofthe experimental agent for 7 days. Oxifulvic acid caused a dose-relatedinhibition of cell growth (FIG. 1).

The Effects of Oxifulvic Acid on Interleukin 2 (IL-2) Production byHuman Lymphocytes

Phytohemaglutinin (PHA)-stimulated human lymphocyte cultures weretreated with oxifulvic acid at 60, 80 and 100 μg/ml for 2 days,whereafter the cells were centrifuged and the supernatant assayed forIL-2 levels, using a Biotrak TM human interleukin 2 ELISA system fromAmersham TM (Amersham International Plc, Buckinghamshire, England).

Oxifulvic acid caused a statistically significant decrease in IL-2production by stimulated lymphocytes at all three concentrations tested(FIG. 2).

Evaluation of Oxifulvic Acid as an Anti-Inflammatory Compound In Vivo

1. Evaluation in Dinitrofluorobenzene Sensitized Mice

The effects of oxifulvic acid (4.5 and 9% cream applied topically) in2,4-dinitro-1-fluorobenzene (DNFB) sensitized mice were evaluated using50 BALB C female mice (6-7 weeks old) according to the method describedby Rheins et al (1987). The mice were sensitized by application of DNFBto the shaved abdomen, divided into 5 groups and challenged on the rightear 6 days later. The inflamed ears of the mice in each group weretreated topically for two days with one of the following creams i.e.control cream, oxifulvic acid 4.5%, oxifulvic acid 9%, dichlophenacsodium 1% (Voltaren Emulgel®) and betamethasone 0.1% (Betnovate®). Thethickness of the ear was measured with a clock gauge before thechallenge and 24 and 48 h later.

All four treatments caused a significant decrease in inflammation onboth days (FIG. 3). These results were confirmed microscopically aftertermination of the mice and embedding the affected ears in paraffin wax.

No signs of toxicity was observed during the two days of treatment withthe two oxifulvic acid creams.

An experiment was also carried out to study the possible variationbetween creams derived from oxifulvic acid batches sampled fromdifferent runs (98100055 and 98110067) as well as from different batches(99030086 to 99030089) sampled on consecutive days from the samecontinuous run. In this study 7 groups of 5 mice per group were used.The first group was treated with control cream whereas the other 6groups were treated with the above-described 6 different 9% oxifulvicacid creams. The results of this experiment are reflected in FIG. 4.

Again no sign of toxicity was observed during the two days of treatmentwith the six oxifulvic acid creams.

Similar results were obtained as in the previous experiment. Oxifulvicacid, once again, caused a statistically significant inhibition ofinflammation in this model. An analysis of variance (two-way ANOVA) wasperformed on the results obtained with the different oxifulvic acidcreams. For day one the P value is 0.131 and day two 0.761. There was,therefore, no significant difference between the various samples tested.

2(a) Evaluation in Pyotraumatic Dermatitis in Cats and Dogs

This trial was done by Dr O J Botha, Hatfield Bird and Animal Hospital,Pretoria. Ten clinical cases were entered in the trial. To qualify forthis trial the following entities had to be present on the skin of theanimals: wheal, erythema, pruritis and pyogenesis. Lesions wereclassified as slight, mild or severe before treatment. No othertreatment was allowed during the trial. Owners were supplied with the 9%oxifulvic acid cream and instructed to apply the cream twice a day tothe affected areas. This treatment had to continue for seven days. Theowners were asked to return exactly seven days after the treatmentcommenced and the cases were reevaluated clinically, biopsied andphotographed.

Decreased inflammation was observed clinically as well as pathologicallyin all 10 cases studied as can be seen from the histopathologicalinvestigation described hereinafter. No side effects were noted in anyof the cases. In most cases it was noted that resolutions of the lesionswere complete and did not recur. Only in one of the cases, described asa chronic and longstanding case, did the owner return after 3 daysbecause the dog was still scratching extensively. A short-actingcortisone was then administered and the dog responded favourably. Atypical example of the results obtained during this trial can be seen inFIG. 5. These photographs were taken before and after the treatment of adalmation dog with lesions on the left medial humerus. Before treatment(FIG. 5A) severe wheal, erythema and pyogenesis were noted while aftertreatment (FIG. 5B) an absence of erythema and pyogenesis and only veryslight remaining wheal were noted. In addition, the biopsy lesions ofthe first biopsy were completely healed.

2(b) Histopathological Investigation: Skin Biopsies: Clinical Study:Topical Ointment—9% Fulvic Acid Cream

Method

Clinical cases having lesions of allergic dermatitis and wet eczema wereidentified in different species of animal as shown in Table 3.

TABLE 3 Information on clinical cases Lab no Treatment group Species3973 A1 Control Cat (house cat) 3973 A2 Treatment Cat (house cat) 3973B1 Control Dog (Bull Mastiff) 3973 B2 Treatment Dog (Bull Mastiff) 3973C1 Control Dog (Bull dog) 3973 C2 Treatment Dog (Bull dog) 4991 A1Control Dog (Dalmation) 4991 A2 Treatment Dog (Dalmation) 4991 B1Control Dog (Maltese) 4991 B2 Treatment Dog (Maltese) 4991 C1 ControlDog (St. Bernard) 4991 C2 Treatment Dog (St. Bernard) 4991 D1 ControlDog (Maltese) 4991 D2 Treatment Dog (Maltese)

Biopsies of control lesions (prior to treatment) of approximately 3 mmwere collected, fixed in formalin and followed up with biopsies as closeas possible to the site of the previous biopsy following treatment.

The formalin-fixed skin-biopsies were processed according to standardroutine methods used for histological studies, embedded in paraffin-waxand slices of 6 μm each were prepared. All slices were coloured usingthe Haemotoxillin and Eosine staining method.

Histopathological Findings

The histopathological findings are shown in Table 4. The control sampleswere initially investigated for specific morphological lesions whichwere graded as indicated in Table 4. The biopsies of the treated lesionswere evaluated similarly.

It must be kept in mind that in each case each lesion could only becompared to its original control lesion, due to the fact that thespecific ethiology and morphological changes differed in each of thecases.

TABLE 4 Histopathological findings: Clinical trials: Enerkom Case 3973A1: Control lesion Surface exudative dermatitis (3+) Ulceration (3+)Dermal inflammation (2+) Case 3973 A2: Treated lesion Surface exudativedermatitis (−) Ulceration (−) Dermal inflammation (1+) Case 3973 B1:Control lesion Hyperplastic epithelia (3+) Hyperpigmentation (2+) Dermalinflammation (3+) Case 3973 B2: Treated lesion Hyperplastic epithelia(−) Hyperpigmentation (1+) Dermal inflammation (1+) Case 3973 C1:Control lesion Surface exudation (3+) Hyperplastic epidermis(3+) Dermalinflammation (3+) Case 3973 C2: Treated lesion Surface exudation (−)Hyperplastic epidermis (3+) Dermal inflammation (1+) Case 4991 A1:Control lesion Ulceration and exudation (2+) Hyperplastic epidermis (2+)Dermal inflammation (2+) Case 4991 A2: Treated lesion Ulceration andexudation (−) Hyperplastic epidermis (1+) Dermal inflammation (1+) Case4991 B1: Control lesion Surface exudation (2+) Hyperplastic epidermis(2+) Dermal inflammation (2+) Case 4991 B2: Treated lesion Surfaceexudation (−) Hyperplastic epidermis (2+) Dermal inflammation (2+) Case4991 C1: Control lesion Surface exudative dermatitis (3+) Ulcerativeboils (2+) Hyperplastic epidermis (2+) Dermal inflammation (3+) Case4991 C2: Treated lesion Surface exudative dermatitis (−) Ulcerativeboils (−) Hyperplastic epidermis (3+) Dermal inflammation (1+) Case 4991D1: Control lesion Hyperplastic epidermis (3+) Dermal inflammation (3+)Case 4991 D2: Treated lesion Hyperplastic epidermis (3+) Dermalinflammation (1+) Key: − absent 1+ light 2+ medium 3+ severe

Conclusion

These results must be interpreted as being histopathologicalobservations only. These results are presented in addition to theclinical observations of the lesions that were treated.

In general, and with due regard to the abovementioned limitations, itappears as if healing was promoted and inflammation reduced in thetreated lesions.

The surface (shallow) exudative dermatitis (acute inflammation) did showaccelerated healing in all cases. Hyperplastic epidermis (acantosis) isa chronic condition and thus showed little change upon treatment. Thedegree of dermal inflammation was also reduced in all except one of thetreated lesions when compared to the original untreated lesions.

There were no controls or lesions that were left untreated for the sameperiod of time as the treated lesions. Spontaneous healing can thus notbe excluded as a contributing factor when interpreting these results.

Toxicity Studies in Experimental Animals

The applicant has undertaken acute and chronic toxicity studies whichserved to prove the safety and very low toxicity of oxifulvic acid.

Acute oral and dermal toxicity studies, acute dermal and eye irritationstudies, skin sensitization studies as well as subchronic oral anddermal toxicity studies, in which the oxifulvic acid solution (25.4%concentrate) and the oxifulvic acid cream (formulated to contain 5.33%oxifulvic acid in this particular instance) were evaluated. The resultsof the investigations are summarized in Tables 5 and 6.

TABLE 5 25,4% Oxifulvic acid toxicity studies STUDY NUMBER TYPE OF STUDYRESULTS 1388 Acute oral toxicity in rats LD₅₀ value >3380 mg/kg 1389Acute dermal toxicity in rats LD₅₀ value >5712 mg/kg 1390 Acute dermalirritation Non-irritant 1391 Acute eye irritation Non-irritant 1392 Skinsensitization Weak skin sensitizer 1520 Subchronic oral toxicity inHighest dose tested was rats (90 day study) 1000 mg/kg/day. No deaths orsick animals. Slight alterations in blood chemistry and slight decreasesin body masses. 1530 Subchronic dermal toxicity in Highest dose testedwas rats (90 day study) 1000 mg/kg/day (of the active substance) Nodeaths or sick animals. Slight alterations in blood chemistry and slightdecreases in body masses.

TABLE 6 5,33% Oxifulvic acid cream toxicity studies STUDY NUMBER TYPE OFSTUDY RESULTS 1393 Acute oral toxicity in rats LD₅₀ value >4147 mg/kg1394 Acute dermal toxicity in rats LD₅₀ value >8599 mg/kg 1395 Acutedermal irritation in Slight irritant rabbits 1396 Acute eye irritationin rabbits Non-irritant 1397 Skin sensitization in guinea Moderate skinsensitizer pigs 1512 Subchronic dermal toxicity in Highest dose testedwas rats (90 day study) 1000 mg/kg/day. No deaths or sick animals.Slight alterations in blood chemistry and local irritation at site ofapplication

Discussion

The data shows that none of the products produced any measurabletoxicity during the acute dermal or oral exposure tests.

During the sub-chronic oral and dermal toxicity studies with bothsubstances very high doses were used. Animals were dosed with 1000mg/kg/day of the active substance in the oxifulvic acid study. The 5.33%oxifulvic acid cream was applied to the skin of rats at a dose rate of1000 mg/kg/day for a period of 90 days. No abnormal clinical signs werenoticed and none of the animals died during the studies. The changes inthe clinical pathology and body masses in the animals receiving the testitem were relatively small.

The Antimicrobial Properties of Oxifulvic Acid

The antimicrobial properties of an oxifulvic acid solution (25.4 percentby mass of the fulvic acid) and a 4.5 percent by mass oxifulvic acidcream were evaluated in vitro on a number of well known pathogens. Theresults obtained are presented in Tables 7 and 8. The symbol + denotesgrowth, and the symbol − denotes no growth.

TABLE 7 Antimicrobial activity of 25.4% oxifulvic acid solution DilutionOrganism 1:2 1:10 1:20 1:40 β-Hemolytic streptococcus − − − +Streptococcus faecalis − − − + Klebsiella pneumoniae − − − + Pseudomonasaeruginosa − − − − Candida spp. − − − + Escherichia coli − − + + Proteusmirabilis − − − + Staphylococcus aureus − − + +

TABLE 8 Antimicrobial activity of 4,5% oxifulvic acid cream DilutionOrganism 1:2 1:10 1:20 β-Hemolytic streptococcus − + + Streptococcusfaecalis − + + Klebsiella pneumoniae − + + Pseudomonas aeruginosa − + +Candida spp. − + + Escherichia coli − + + Proteus mirabilis − + +Staphylococcus aureus − + +

Further, the efficacy of the 4.5 percent oxifulvic acid cream and a 25.4percent by mass oxifulvic acid solution to inhibit the growth ofbacteria and fungi was tested in accordance with the SABS method 730 (invitro) on a few test organisms. The results obtained are set out inTable 9.

TABLE 9 Bacteriostatic and fungistatic efficacy of 4.5% oxifulvic acidcream and 25.4% oxifulvic acid solution Diameters (mm) of the zones ofinhibition Pseudomonas Escherichia Staphylococcus Aspergillus CandidaStreptococcus Samples Dilution aeruginosa coli ureus niger albicanspyogenes Solution 25.4% 29.4 29.36 >29.0 18.83 118.36 22.71 Solution5000 ppm N/T N/T N/T No zone No zone N/T Solution 1000 ppm No zone Nozone No zone N/T N/T N/T Solution 1500 ppm No zone No zone No zone N/TN/T N/T Cream  4.5% 15.6 14.74 28.8 9.66  9.58 13.39 N/T = not tested

From the aforegoing studies it can be seen that oxifulvic acid exhibitssome measure of antimicrobial activity or bacteriostatic or fungistaticefficacy against some of the test organisms, even when formulated in acream.

The 4.5 percent oxifulvic acid cream was subjected to a preservativeefficacy test by the Microbiological Division of the SABS and it wasfound that the cream complied with the requirements of the USP 23(1995).

Antiviral Activity of Oxifulvic Acid

Methods

The following viruses were tested:

Human herpes simplex virus type 1

Human adenovirus type 2

Simian rotavirus SA 11

Poliovirus type 1 Sabin vaccine strain

Coxsackie B virus type 1 laboratory strain

Coxsackie A virus type 9 laboratory isolate

Viruses were grown in either vervet monkey kidney cells or a primaryliver cancer cell line (PLC/PRF/5). Stock virus suspensions weretitrated to establish their titre and to prepare a viral suspensioncontaining 100×50% tissue culture infectious dose (100 TCID₅₀)/200 μl.

Prevention of binding of viruses to the host cell: Monolayers of theappropriate cell cultures in 96-well microtitre trays were washed andstarved for a minimum of 1 hr in serum-free MEM. After starvation,doubling dilutions of oxifulvic acid in serum-free MEM were added toeach well together with 100 TCID₅₀ virus. The microtitre trays wereincubated at 37° C. and examined daily, for 7 days, for CPE. Wellsinoculated with 100 TCID₅₀ viruses and no oxifulvic acid acted aspositive controls. The appearance of CPE indicated that no inhibition ofbinding had taken place.

Inhibition of viral replication in the host cell: Monolayers of theappropriate cell cultures in 96-well microtitre trays were washed andstarved for a minimum of 1 hr in serum-free MEM. After starvation 100TCID₅₀ virus was added to all wells and allowed to adsorb for 1-2 hrs.After adsorption, the unadsorbed viruses were washed from the wellsusing serum-free MEM. Thereafter doubling dilutions of oxifulvic acid,in serum-free MEM, were added to the appropriate wells. The microtitretrays were incubated at 37° C. and examined daily, for 7 days, for CPE.Wells inoculated with 100 TCID₅₀ viruses and no oxifulvic acid acted aspositive controls. The appearance of CPE indicated that no inhibition ofviral replication had taken place.

Results

The effects of oxifulvic acid on the binding of viruses to the hostcells as well as on the replication of viruses can be seen in Tables 10Aand 10B.

TABLE 10A Effects of oxifulvic acid on the binding of viruses to thehost cells Lowest concentration Lowest concentration at at which limitedwhich 100% viral inhibition viral inhibition was noted at day 6 or 7post was notices Virus infection (mg/ml) (mg/ml) Herpes simplex virus1.875 0.234 type 1 Human adenovirus type 2 3.75 0.937 Simian rotavirusSA 11 1.875 0.058 Polio virus type 1 1.875 0.468 Coxsackie B virus type1 1.875 0.937 Coxsackie A virus type 9 1.875 1.875 TABLE 10B Effects ofoxifulvic acid on the replication of viruses Lowest Lowest concentrationat concentration which 100% viral inhibition at which limited was notedat day 6 or 7 post viral inhibition Virus infection was noticed Herpessimplex virus 1.875 0.937 type 1 Human adenovirus type 2 3.75 1.875Simian rotavirus SA 11 3.75 0.234 Polio virus type 1 0.468 0.234Coxsackie B virus type 1 1.875 0.937 Coxsackie A virus type 9 1.8750.468

Discussion

Oxifulvic acid prevented the binding of the six experimental viruscultures between 1.87 mg/ml and 3.75 mg/ml whereas inhibition of viralreplication was inhibited at concentrations between 0.469 mg/ml and 3.75mg/ml. Limited inhibition of viral replication was noted at aconcentration as low as 0.103 mg/ml in the case of simian rotavirus SA11.

Oxifulvic acid is therefore a compound that

i) scavenges the neutrophil-derived pro-inflammatory reactive oxidantsuperoxide;

ii) decreases production of the lymphocyte-derived pro-inflammatorycytokine IL-2;

iii) inhibits a mixed lymphocyte reaction typical of a transplantedorgan rejection reaction;

iv) has in vitro activity against Gram positive, Gram negative, as wellas fungal and viral human pathogens;

v) inhibits a contact hypersensitivity reaction induced indinitrofluorobenzene sensitized mice as effectively as generally usedanti-inflammatory agents such as dichlophenac sodium and betamethasone(these two agents are however associated with serious adverse sideeffects);

vi) has in vivo anti-inflammatory activity against pyotraumaticdermatitis in cats and dogs; and

vii) has very low toxicity in experimental animals.

REFERENCES

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What is claimed is:
 1. A method of treating a subject presentinginflammation, acne, eczema, a bacterial infection, a viral infection, orany combination thereof, comprising administering to said subject acomposition comprising fulvic acid, or a salt, ester or derivativethereof.
 2. The method of claim 1 in which the composition isadministered orally or topically.
 3. The method of claim 1 in which thefulvic acid, or salt, ester or derivative thereof is oxifulvic acid or asalt, ester or derivative thereof.
 4. The method of claim 2 in which thefulvic acid, or salt, ester or derivative thereof is oxifulvic acid or asalt, ester or derivative thereof.
 5. The method of claim 3 in which theoxifulvic acid, or salt, ester or derivative thereof comprises 11.5 to15.5 milliequivalents/g of total acid groups, 8.5 to 12.5milliequivalents/g of carboxylic acid groups and 2.3 to 3.7milliequivalents/g of phenolic groups.
 6. The method of claim 4 in whichthe oxifulvic acid, or salt, ester or derivative thereof comprises 11.5to 15.5 milliequivalents/g of total acid groups, 8.5 to 12.5milliequivalents/g of carboxylic acid groups and 2.3 to 3.7milliequivalents/g of phenolic groups.
 7. The method of any one ofclaims 1-6 in which the subject is a human.
 8. The method of any one ofclaims 1-6 in which the subject is a non-human animal.